A significant number of the adult population world-wide is affected by periodontal diseases. These chronic biofilm infections of the tooth-supporting tissues that are the major cause of tooth loss have also been associated with systemic diseases. Even though extensive inventory of the oral flora and general structural information about oral biofilms are available, detailed analysis of the species distribution and their interactions in biofilms causing periodontal disease is largely missing. The long-term objective of this project is to establish fluorescence in situ hybridisation (FISH) for the detailed examination of important subgingival biofilm features, including species presence and abundance, localization pattern, biofilm-specific morphotype, the role of as-yet-uncultured organisms and indentification of bacteria with pivotal role in biofilm structure formation. FISH is chosen as a tool since this versatile technique enables culture independent detection of microorganisms on all taxonomic levels and visualisation of their distribution in undisturbed samples. Furthermore, the role of specific microorganisms in architectoral biofilm aspects as well as their interaction will be examined on a quantitative level. In compliance with the mission of the NIDCR, this will provide important information for the development of new diagnostic and medical targets in the treatment of periodontal diseases which are still difficult to cure due to the persistent nature of the infection. In this specific application we propose to employ FISH to determine proportion, spatial distribution and co- localisation pattern of selected putative periodontal pathogens within the biofilm community. This method will be applied in combination with a carrier-based in vivo model system that allows sampling of naturally grown periodontal biofilms. These biofilms will be examined for the presence of suspected oral pathogens (e.g. oral treponemes, Fusobacterium ssp., Porphyromonas gingivalis, Tannerella forsythia, Filifactor alocis, TM7 complex) using specific FISH probes. Microscopic examination of whole and sectioned carriers will enable qualitative evaluation of their respective abundance, spatial distribution and co-localisation (indicative for interactions between microorganisms). To enable quanitative analysis of the data obtained, the novel digital image analysis program daime that was optimized for FISH labeled samples will be employed. This study will validate current ideas of periodontal disease development and provide detailed information about the bacteria involved including those that cannot be studied in laboratory test tube cultures. Knowing which bacteria play central roles in the build-up of the biofilm communities in periodontitis could identify new marker organisms for diagnostic tests. This could lead to treatments in which only key harmful organisms will be eliminated compared to the "kill all" approaches of existing therapies in which beneficial bacteria are not spared. Furthermore, failed treatments and treatments outcomes could be easily monitored. Project Narrative: Chronic bacterial infections of the tooth-supporting tissue (gum and bone), the periodontal diseases affect a significant portion of adults. It is the major cause for tooth loss and may be related to so-called systemic conditions such as heart disease. Periodontal diseases are difficult to cure permanently. Repetitive treatments are necessary to keep these infections in check since their cause, highly structured bacterial communities and yet unknown bacteria that reside within the periodontal pockets are difficult to remove completely. Our research will help to understand which type of bacteria are relevant for formation of these anchoring structures that keep other bacteria in place, who is most abundant and which types of bacteria may interact with each other to promote disease. If successful, this study will provide useful information for the development of new diagnostic and therapeutic tools.